All eukaryotes have an essential organelle, the proteasome, for degrading misfolded and unneeded cellular proteins. Proteins are targeted for degradation by the posttranslational modification of a polyubiquitin tag, in which isopeptide linkages connect a lysine side chain on ubiquitin or on the target protein to the C-terminus of the distal ubiquitin. Tetraubiquitin chains linked through Lys48 are potent degradation signals, and recent studies indicate that some heteropolymers also constitute a degradation signal. The effects of most connectivities are not fully characterized, however, and existing methodologies cannot access useful quantities of all potential, distinct polyubiquitin tags. Traceless Staudinger Ligation is a chemical method for concatenating polypeptides. The Raines lab recently designed a procedure for running this reaction in aqueous buffer at physiological pH. This methodology shall be further optimized for folded, functional proteins, and used to generate ubiquitin polymers of defined length and connectivity. Branched polymers shall also be generated;branched polyubiquitin chains appear to inhibit the proteasome, but characterization of this effect is incomplete. The well-characterized protein Sid shall be used as the degradation target. Sid p shall be tagged with a variety of tetraubiquitin homopolymers and heteropolymers. Active proteasomes shall be isolated from yeast, and used in degradation assays with the purified Sid p targets. Ultimately, this will enable the "code" for what polyubiquitin connectivities constitute a degradation signal to be determined. Furthermore, this chemical tagging method will enable the limits of proteasomal degradation to be probed. The proteasome is capable of selectively degrading one protein in a multi-protein complex. Polyubiquitination of an unnatural target will challenge the proteasome to separate and degrade one target of two extremely tightly-bound partners. The protein RNase 1 shall be used as the degradation target, for it has an unusually stable interaction (an 81-day half-life) with the protein RI (RNase Inhibitor). PUBLIC HEALTH RELEVANCE: The malfunction of the ubiquitin-mediated protein-degradation pathway is correlated with a variety of human diseases, including multiple types of cancer and many neurological disorders. The further- understanding of the functions (and malfunctions) that occur within this pathway is therefore crucial to the development of drugs and finding treatments for these diseases.